2,19-methyleneoxy and 2,19-methylenethio bridged steroids as aromatase, 19-hydroxylaser inhibitors and methods of their use in the treatment of estrogen mediated disorders

ABSTRACT

The present invention is directed to a method of using a certain compounds which are 2,19-methyleneoxy or 2,19 methylenethio bridged steroids, and related steroidal compounds as inhibitors of the enzyme steroid aromarase, 19-hydroxylase and as treatment for various estrogen dependent/mediated disorders including hormonal dependent breast cancer.

The present application is a continuation-in-part of application Ser.No. 08/049,786 filed Apr. 19, 1993 which is a continuation-in-part ofapplication Ser. No. 07/803,239, filed Dec. 5, 1991 and now abandoned;which is division of application Ser. No. 07/674,640, filed Mar. 25,1991 now U.S. Pat. No. 5,099,037; which is continuation-in-part ofapplication Ser. No. 07/453,441, filed Dec. 20, 1989 and now abandoned.

BACKGROUND OF THE INVENTION

The estrogen hormones, estrone and esitradiol, are involved in manyphysiological processes. The formation of these steroids is regulated bya number of enzymes. The enzyme aromatase is the rate limiting enzyme inthe non-reversible conversion of the androgen hormones, testosterone andandrostenedione, to the estrogen hormones, estradiol and estrone.Compounds such as aromarase inhibitors may thus regulate or inhibitandrogen to estrogen conversion, and have therapeutic utility intreating clinical conditions potentiated by the presence of estrogens.

19-Nordeoxycorticosterone (19-norDOC) is known to inducemineralocorticoid hypertension. In the biosynthetic formation of19-norsteroids, such as 19-norDOC, the initial step is the adrenalhydroxylation of an appropriate steroid such as deoxycorticosterone(DOC). The inhibition of the biosynthetic formation of 19-norDOC byinhibition of 19-hydroxylation of DOC would thus serve to decrease thelevel of 19-norDOC present in the animal involved and reducehypertensive effects tributable to the presence of this material.

SUMMARY OF THE INVENTION

The present invention is directed to 2,19-bridged steroidal aromataseand 19-hydroxylase inhibitor compounds, their related intermediates, aprocess for their preparation, and their use in the treatment of variousestrogen dependent/mediated disorders. These compounds may berepresented by the following formulas: ##STR1## wherein

----- represents a single or double bond,

A is O, S, SO, or SO₂,

R is H, ═CH₂, ═O, or --OH,

R¹ is H or C₁₋₄ alkyl,

R² is ═O, --OH, or --O--(C₁₋₄ alkanoyl),

X is ═O, ═CH₂, --OH, or --O--(C₁₋₄ alkanoyl), and

Y is H, --OH, or --O--(C₁₋₄ alkanoyl), and when Y═H, OH, or --O--(C₁₋₄alkanoyl), X may not include --OH, and R may not include ═O or --OH.

Examples of the alkyl groups referred to above are methyl, ethyl andpropyl. Examples of the alkanoyl groups referred to above are acetyl,propionyl and butyryl. The double bonds, as represented by the dottedlines above are selected in such a way tat the compounds must contain atleast one double bond, usually in the A-ring of the standard steroidskeleton, although it can also be located at the 5,6-position in theB-ring. If the double bond is located at the 5,6-position, then theother dotted lines represent single bonds. When the system is doublyunsaturated, the double bonds are located at the 4,5- and the6,7-positions.

Since the compounds of the present invention can be considered ascontaining a bridged steroid structure, it is possible to name them asderivatives of the basic steroid involved. When this is done with theoxygen-bridged compounds of the present invention, the compounds can bereferred to as 2,19-(methylenoxy) steroids. This indicates that a --CH₂O-- group connects the 2- and 19-positions with the carbon attached onthe β-side of the 2-position and the oxygen attached to the 19-carbonatom. The sulfur bridged compounds can be described in a similar way.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the present invention are inhibitors of aromarase and19-hydroxylase. As aromatase inhibitors, they are useful in treatinghyperestrogenemia. The compounds are useful in controlling abnormallyhigh levels of estrogens, both when the high levels observed arerelatively steady, or when there are brief surges of elevated levelsoccurring as part of cyclical body functions. Both females and males canbe treated, although obviously, the level of estrogens which would beconsidered high in males would be much lower than the amount consideredhigh in females.

These compounds are also useful as anti-fertility agents to preventovulation or implantation in females, or to reduce the mating behaviorin males where brain aromatization is required for such behavior. Thesecompounds further have value in treating diseases of the male, such asgynecomastia, male infertility resulting from elevated estrogen levels,and hyperestrogenemia, which may precede myocardial infarction. Othermale diseases for which the compounds of this invention are applicableinclude the therapeutic and/or prophylactic treatment of prostaticdiseases, including prostatic hyperplasia, a disease of the estrogendependent stromal tissue, and prostatic cancer.

There is substantial clinical evidence to indicate that many tumor typesare associated with elevated estrogen production. Ovariectomy,adrenalectomy and hypophysectomy are commonly employed in patients withbreast cancer as a means of reducing the amount o estrogen. Non-surgicalprocedures include treatments with high levels of steroids,anti-estrogens and inhibitors of steroidal enzymatic pathways. Treatmentwith antiestrogens results in about one-third of the patients obtainingobjective tumor regressions. Andrenalectomy will cause regression ofbreast cancer in postmenopausal women with hormonal-dependent tumors,presumably as the result of reduction in available estrogen derived fromandrostenedione, whose source is primarily from the adrenals. Growth ofseveral lines of breast cancer cells have been shown to be estrogendependent or at least estrogen mediated, and can be inhibited, bycompounds which antagonize estrogen action.

Breast tumors excised from postmenopausal women contain estradiolconcentrations that are 5-50 fold higher than plasma estrogen levels.The aromatase activities of breast tumors are associated with stromalfibroblasts and adipose tissue. Fibroblast and adipose tissue aromataseactivity is stimulated by a variety of promoters such asglucocorticoids, phorbal esters, cytokines and growth factors that canact through associated receptors via autocrine and paracrine pathways topromote tumor growth.

The aromarase inhibitors of the present invention can effectivelyprevent the biologically active estrogens from reaching endocrine tumorsor reduce estrogen biosynthesis in those tumors capable of endogenousestrogen synthesis, thereby producing remissions of hormonal dependentbreast cancer. Thus, these compounds are useful in the treatment ofbreast, ovarian, uterine and pancreatic tumors as well as diseasecondition such as galactor rhea, McCune-Albright syndrome, benign breastdisease, endometriosis, and polycystic ovarian disease.

The bioconversion of deoxycorticosterone via a 19-hydroxylase pathway to19-nordeoxycorticosterone potentiates its mineralocorticoid activity.Mineralocorticoid excess results in a syndrome characterized byhypokalemia, metabolic alkalosis, polydipsia, polyuria, and hypertensiveconditions. Increased excretion of 19-nordeoxycorticosterone has beenreported for hypertensive patients, including those with primaryaldosteronism Cushing's syndrome, 17β-hydroxylase deficiency, andindividuals with essential hypertension. As 19-hydroxylase inhibitors,these compounds may be useful as antihypertensive agents and formanagement of edemous conditions often associated with sodium retentionand potassium loss.

The compounds of the present invention which have a pregnane side chainare further useful in that a C₁₇₋₂₀ lyase enzyme can cleave theindicated side chain to give the corresponding 17-oxygenated androstenecompounds which, as already indicated, are useful as aromataseinhibitors.

To achieve their desired effect, the compounds of the present inventionmay be administered orally, parenterally, for example, intravenously,intraperitoneally, intramuscularly, or subcutaneously, including theinjection of the active ingredient directly into tissue or tumor sites,to a patient in need of treatment. The term patient is taken to mean awarm-blooded animal, for example, mammals such as humans, primates,cattle, dogs, cats, horses, sheep, mice, rats, and pigs. These compoundsmay also be administered in the form of a pharmaceutical preparation,and may further be incorporated into sustained delivery devices. Theamount of compound administered will vary over a wide range and be anyeffective amount. Depending on the patient to be treated, the conditionto be treated, and mode of administration, the effective amount ofcompound administered will vary from about 0.01 to 150 mg/kg of bodyweight per day, and preferably from about 0.1 to 50 mg/kg body weightper day.

For oral administration, the compounds can be formulated into solid orliquid preparations, such as capsules, pills, tablets, troches, powders,solutions, suspensions, or emulsions. The solid unit dosage forms can bea capsule which can be of the ordinary gelatin type containing theactive compound and a carrier, for example, lubricants and inert fillersuch a lactose, sucrose and corn starch. In another embodiment, anactive compound of the invention can be tableted with conventionaltablet bases such as lactose, sucrose and corn starch in combinationwith binders such as acacia, corn starch, or gelatin, disintegratingagents such as potato starch, or alginic acids and a lubricant such asstearic acid or magnesium stearate.

For parenteral administration the compounds may be administered asinjectable dosages of a solution or suspension of the compound in aphysiologically acceptable diluent with a pharmaceutical carrier whichcan be a sterile liquid such as water-in-oil with or without theaddition of a surfactant and other pharmaceutically acceptableadjuvants. Illustrative of oils which can be employed in thesepreparations are those of petroleum, animal, vegetable or syntheticorigin, for example, peanut oil, soybean oil, and mineral oil. Ingeneral, water, saline, aqueous dextrose and related sugar solutions,alcohols and glycols, such as propylene glycol or polyethylene glycolare preferred liquid carriers, particularly for injectable solutions.

The compounds can be administered in the form of a cutaneous patch, adepot injection, or implant preparation which can be formulated in sucha manner as to permit a sustained release of the active ingredient Theactive ingredient can be compressed into pellets or small cylinders andimplanted subcutaneously or intramuscularly as depot injections orimplants. Implants may employ inert materials such as biodegradablepolymers and synthetic silicones, for example, Silastic®, siliconerubber manufactured by Dow Corning Corporation. Further information onsuitable pharmaceutical carriers and formulation techniques are found instandard texts such as Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Pa.

Inhibition of aromatase activity is demonstrated by using laboratorymethods similar to procedures described in U.S. Pat. No. 4,322,416, andas published in Johnston et al., Endocrinology 115:776, 1984, andBurkhart et al., Steroids 45:357, 1985.

In this assay, the inhibitor is preincubated with enzyme prior toassaying for activity in the presence of high substrate levels. Atime-related decrease in enzyme activity can be indicative ofirreversible binding of the inhibitor with the enzyme.

In the time-dependent assay, an amount of the enzyme inhibitor in 100 μlof the assay buffer described above which will provide assayconcentrations which are usually between 1 nM and 10 μM are added to 35ml centrifuge tubes containing 600 μl of the NADPH generating system.The preincubation is started by the addition of 700 μl of aromarasepreparation, usually 500-800 μg of microsomal protein per ml of assaybuffer. These preparations are mixed using a vortex mixer and incubatedfor 0, 10, 20, or 40 minutes at 25° C. Then 100 μl of androstenedione([6.8 μM) containing 1β-³ H androstenedione is added in assay buffer toprovide an assay concentration of substrate (0.55 μM) which is at leastten times k_(m) of androstenedione (0.04 μM). Following vortexing, theenzyme incubation is continued for 10 minutes before being terminated bythe addition of chloroform. The amount of radioactivity in the aqueousfraction is determined by scintillation procedures. The enzymaticactivity for each concentration of inhibitor at each time period ofpreincubation is calculated as a percent of the " 0" minute vehiclecontrol arbitrarily set at 100%. Therefore, the present enzymeinhibition is expressed as a percentage: (100 percent minus percentenzyme activity with inhibitor present).

Enzyme kinetic analysis utilized Kitz-Wilson plots for time-dependentassays. These analyses provide estimates of apparent K_(i) ofinactivation which represents the inhibitor concentration required toproduce half-maximal rate of enzyme inactivation. The pseudo first-orderrate constant for enzyme inactivation (k_(cat)) and the half-time ofinactivation (τ₅₀) of infinite inhibitor concentrations were determined.The ratio of k_(cat) /^(K) i (inactivation) provides an index numberwhich increases with increased efficiency of enzyme inactivation andincreased inhibitor affinity for the enzyme active site. When tested bythis procedure, the following results were observed for compounds ofthis invention:

2,19-(methyleneoxy)androst-4-ene-3,17-dione, also named[3R-(3α,6aα,6bα,8aβ,11aα,11bβ)]-3,4,6b,7,8,8a,10,11,11a,11b,12,13-dodecahydro-8a-methyl-6H-3,6a-methanocyclopenta[5,6]naphth[1,2-c]oxocin-2,9-dione[Compound (4) below]: K_(i) (nM)=17.6; τ₅₀ (min)=2.86; k_(cat) /K_(i)=227,300.

2,19-(methylenethio)androst-4-ene-3,17-dione, also named[3R-(3α,6aα,6bα,8aβ,11aα,11bβ)]-3,4,6b,7,8,8a,10,11,11a,11b,12,13-dodecahydro-8a-methyl-6H-3,6a-methanocyclopenta[5,6]naphtho[1,2-c]thiocin-2,9-dione[Example 3, last paragraph]: K_(i) (nM)=53.0; τ50 (min)=1.65; k_(cat)/K_(i) =132,103.

2,19-(methyleneoxy)androsta-4,6-diene-3,17-dione, also named[3R-(3α,6aα,6bα,8aβ,11aα,11bβ)]-3,4,6b,7,8,8a, 10,11,11a,11b-decahydro-8a-methyl-6H-3,6a-methanocyclopenta[5,6]-naphth[1,2-c]oxocin-2,9-dione [Compound (15) below]: K_(i)(nM)=21.6; τ50 (min)=2.82; k_(cat) /Ki_(i) =189,658.

When assaying compounds for 19-hydroxylase inhibiting activity,compounds were solubilized in dimethyl sulfoxide (DMSO) at 10 mM anddiluted in DMSO to provide 0.01-10 μM final concentration when 2 μLaliquots were added to microcentrifuge assay tubes. Assay buffer (10 mMKCl, 1 mM EDTA, 100 mM Tris-HCl at pH 8.0) which had been supplementedwith an NADPH-generating system to provide assay concentrations of 1 mMNADPH, 3 mM glucose-6-phosphate and 1 I.U./ml glucose-6-phosphatedehydrogenase were incubated at 37° C. for 5 minutes prior to additionof hamster adrenal mitochondrial protein. Aliquots (180 μL) of thislatter preparation containing 5.1 μg enzyme protein were assayed at 37°C. for 5 minutes following the initiation of the assay by the additionof 20 μL of assay buffer containing radio-labeled DOC (0.85 μM finalconcentration, 0.01 μCi with 99.8% radiochemical purity, NEN ResearchProducts, Boston, Mass.). Assays were quenched by the addition of 800 μLof 20% CH₃ CN-2% HOAc. The reactants were centrifuged for 2 minutes at15,000 xg and analyzed by liquid chromatography (Beckman InstrumentsInc., San Ramon, Calif.) on two C₁₈ Radial Pak columns (Waters,Millipore Corporation, Milford, Mass.) in series (5 μM particles, 0.8×10cm each). Chromatographic buffer A was 10% CH₃ CN-0.1% HOAc and buffer Bwas 80% CH₃ CN-0.1% HOAc. The column was eluted at a flow rate of 1ml/minute with a linear gradient from 0 to 30% buffer B over 36 minutesfollowed 100% buffer B. The amount of remaining labeled DOC substrateand initial hydroxylated products, corticosterone and 19-hydroxy-DOC,were separated and the radioactivity contained in each peak quantitated.The 19-hydroxylase activity was based on the quantity of radiolabeledDOC metabolized, since corticosterone and 19-hydroxy-DOC are theproducts of a single enzyme.

Unlabeled steroids were monitored by their absorbance at 240 nm with aKratus Spectroflow 773 detector (Kratus Analytical Instruments, Ramsey,N.J.). The extinction coefficients for derivatives of DOC were assumedto be similar to that of DOC (ε=17,200 M⁻¹ cm⁻¹). Radioactivity of DOCmetabolites was measured using an online Flow-One scintillationspectrometer (Radiomatic Instrument & Chemical Co., Inc., Tampa, Fla.)with a 1 ml flow cell.

Time-dependent enzyme inhibition was evaluated by preincubating theenzyme with steroidal compound for either 0 or 60 minutes at 37° C.prior to the addition of radiolabeled substrate for a 5 minute assay.Apparent K_(m) for the first hydroxylation of DOC may be estimated bythe double reciprocal plot of Lineweaver-Burk. IC₅₀ 's may begraphically estimated from linear-log plots of enzyme activities and logof inhibitor concentrations.

According to the method of Johnston, J. O. et al., J. Steroid Biochem.,33:521, 1989, which is herein incorporated by reference, the compoundsof the present invention were further evaluated for their effect uponintratumor aromatase activity. Table 1 graphically displays observedeffects of 2,19-(methyleneoxy)-androst-4-ene-3,17-dione on intratumoraromarase synthesis. Compounds that exhibit time-dependent enzymeinhibition which can block intratumoral aromatase activity are preferredas potential therapeutic breast cancer agents.

                  TABLE 1                                                         ______________________________________                                        Inhibition of Intratumor Aromatase Activity:                                  Estrogen formed (picomoles/gram of tumor/hour)                                                    Hours Post treament:                                               Vehicle                                                              Absolute levels                                                               Method   control                                                              % Inhibition                                                                  (3 mg/kg)                                                                              (absolute) 2        4       6                                        ______________________________________                                        intraveneous                                                                           294 ± 5 88 ± 15                                                                             175 ± 12                                                                           186 ± 9                                                   70 ± 5%                                                                             41 ± 4%                                                                            37 ± 3%                               subcutaneous                                                                           121 ± 85                                                                              68 ± 6                                                                              67 ± 3                                                                             78 ± 9                                                    44 ± 5%                                                                              45 ± 32%                                                                          35 ± 7%                               oral     173 ± 9 49 ± 8                                                                              62 ± 7                                                                             64 ± 9                                                    72 ± 5%                                                                             64 ± 4%                                                                            63 ± 5%                               ______________________________________                                    

The compounds of the present invention were also tested for theinhibition of tumor growth. Tumor growth inhibition was tested by thetreatment of 50-day old female rats with the carcinogen,7,12-dimethylbenz[a]anthracene [DMBA] and subsequently evaluated fortumor growth and/or remission (Huggins, C. et al., Nature 189:204, 1961;Russo, J. et al., Lab. Investigation 62:244, 1990). Rats withDMBA-induced mammary tumors (≈300-1300 mm³) were assigned to thefollowing treatment groups: (1) Non-treated controls, (2) animals withsubcutaneous implanted osmotic pump containing vehicle (DMSO/PEG₂₀₀, 1:4v/v), and (3) animals with subcutaneous implanted osmotic pumpcontaining vehicle and 15 mg/kg/day of 2,19-(methyleneoxy)androst-4-ene-3,17-dione. Drug-treated animals also received 15mg/kg/day via subcutaneous injections for the first two days oftreatment as a priming dose. Tumor size was monitored for 42 days ofdrug treatment. Data are presented in Table 2 as the percent of animalswhich exhibited complete tumor regression, partial tumor regression (less than 50% of original tumor volume), no change (50 to 150% oforiginal tumor volume) and tumor progression (greater than 150% oforiginal tumor volume).

                  TABLE 2                                                         ______________________________________                                        DMBA Induced Mammary Tumor Growth                                             Response                                                                              Com-                          Pro-                                            plete   Partial  No change    gressive                                Treatment                                                                             (0%)    (<50%)   (50%≦ × ≦150%)                                                         (>150%)                                 ______________________________________                                        Controls                                                                              6.7     6.7      6.7          80.0                                    2,19-   14.3    42.9     14.3         28.6                                    (methyl-                                                                      eneoxy)-                                                                      androst-                                                                      4-ene-                                                                        3,17-dione                                                                    ______________________________________                                    

Various procedures can be used to prepare the compounds of the presentinvention. Scheme 1 below is used to prepare compound (4),2,19-(methyleneoxy)androst-4-ene-3,17-dione. Alternatively, compound (4)may be named [3R-(3α,6aα,6bα,8aβ,11aα,11bβ)]3,4,6b,7,8,8a,10,11,11a,11b,12,13-dodecahydro-8a-methyl-6H-3,6a-methanocyclopenta[5,6]naphth[1,2-c]oxocin-2,9-dione. To facilitate the understanding of the presentinvention, steroidal nomenclature and numbering are utilized in theprocedures and examples that follow. ##STR2##

Commercially available steroid starting compound (1) is reacted withdiisopropylethylamine and 1-chloro-2,5-dioxahexane to form the compound,19-[(2-methoxyethoxy)methoxy]-androst-4-ene-3,17-dione (2). Thiscompound is then reacted with a mixture of trimethylchlorosilane andlithium diisopropylamide to form the compound19-[(2-methoxyethoxy)methoxy]-3,17-bis[trimethylsilyl)oxy]-androst-2,4,16-triene(3). This compound is then treated with TiCl₄ to give the desiredcompound, 2,19-(methyleneoxy)androst-4-ene-3,17-dione (4).Alternatively, to prepare those compounds wherein A=S, the corresponding19-mercapto steroidal starting compound is utilized, and the reactionproceeds analogous to Scheme 1. The compounds where A=SO and A=SO₂ areprepared from the corresponding compound where A=S by treatment with oneor two equivalents of 3-chloroperoxybenzoic acid, respectively, in asolvent such as methylene chloride.

To prepare the compound bearing the hydroxyacetyl substituent at the17-position (10), Scheme 2 is utilized: ##STR3##

The 2,19-(methyleneoxy)androst-4-ene-3,17-dione (4) is treated with acatalytic amount of acid such as methanesulfonic acid in an excess ofethylene glycol to form the corresponding 3,17-bis(ethylenedioxy)compound (5). This compound is then selectively hydrolyzed at the17-position with 0.15% aqueous perchloric acid in t-butanol anddichloromethane to give the corresponding 17-ketone (6). The ketone isthen reacted with methyl methoxyacetate and lithium diisopropylamidewhereupon the indicated ester (i.e., the methylene group thereof), addsacross the 17ketone to give the 17-substituted 17-hydroxy steroid (7).Dehydration introduces a 17-exocyclic double bond and the resultingmethoxy ester (8) is reduced with a hydride reducing agent such asdiisobutylaluminum hydride to give the corresponding alcohol (9), whichis then further treated with acid to hydrolyze the enol ether and alsothe 3-ketal to give the desired 21-hydroxy-20-keto compound (10).

To prepare compound (14),2,19-(methyleneoxy)androst-4-ene-3,6,17-trione, Scheme 3 is utilized:##STR4##

The diketal starting material (5) is treated with m-chloroperbenzoicacid in dichloromethane at 0° C. to produce the epoxide (11). Theepoxide is opened to the corresponding diol (12) using perchloric acidin THF and H₂ O. The ketals are also removed in this process. The diolis then oxidized to the hydroxy-ketone by Jones oxidation. Thehydroxy-ketone (13) is then taken up in benzene and dehydrated usingp-toluenesulfonic acid to yield the steroidal trione (14).

Compounds containing multiple double bonds on the steroid ring systemcan be obtained by dehydrogenation of the appropriate starting compound.For example, dehydrogenation of2,19-(methyleneoxy)androst-4-ene-3,17-dione (4) with chloranil int-butanol gives the corresponding diene (15) as shown by Scheme 4 below.##STR5##

To obtain compounds of the present invention wherein R is ═CH₂,2,19-(methyleneoxy)androst-4-ene-3,17-dione (4) is reacted with aformaldehyde acetal as shown by Scheme 5 below. ##STR6##

Reagents such as p-toluenesulfonic acid, strong mineral acids, acidicion exchange resin, or preferably, phosphoryl chloride with formaldehydedimethyl or diethyl acetal, are most suitable to effect thiscondensation.

To obtain 2,19-(methyleneoxy)androst-4-ene-3,17-diol (17 ), Scheme 6 isutilized: ##STR7##

The starting compound, 2,19-(methyleneoxy)androst-4-ene-3,17-dione (4 ),is reduced with sodium borohydride in ethanol to yield the correspondingdiol (17). To prepare the 5,6-ene diol (19), the starting compound,2,19-(methyleneoxy)androst-4-ene-3,17-dione (4), is treated with acatalytic amount of p-toluenesulfonic acid and heating in a solvent suchas Ac₂ O. The mixture is then cooled. To this mixture is then addedpyridine followed by ethanol to yield the dienol acetate (18).

Alternatively, the dienol acetate (18) may preferably be prepared byadding an excess of Ac₂ O and a catalytic amount of 70% aqueous HClO₄ tothe steroid (4) in EtOAc. The mixture is then stirred for 15 minutes andpoured into dilute Na₂ CO₃, extracted, and washed with dilute Na₂ CO₃and brine to yield the dienol acetate (18). The dienol acetate (18) isthen treated with calcium borohydride in EtOH at -15° C. The reaction isquenched with HOAc and partitioned between EtOAc and H₂ O to yield thediol (19). Treatment of the diol (19), with an anhydride, such as aceticanhydride, gives the corresponding diacetate.

In another approach to the preparation of the 5,6-ene diol (19), thesteroid (4) is reacted with hexamethyldisilazane in pyridine solutionand trimethylbromosilane to give2,19-(methyleneoxy)-3,17-bis-(trimethylsilyloxy)androsta-3,5,16-trienewhich is then reduced with calcium borohydride in ethanol to give, afterappropriate quenching, the desired product.

To prepare the compound wherein R¹ is CH₃, Scheme 7 is utilized.##STR8##

The known bisketal compound (20) undergoes a Swern oxidation to yieldthe oxidized bisketal (21). This compound is then treated with R¹ MgBror R¹ Li, wherein R¹ is defined above, to produce the R¹ -substitutedhydroxy compound (22). Treatment of (22) with aqueous HCl in THF yieldsthe dione (23). Treatment of the dione (23) in a manner analogous toScheme 1 yields the R¹ -substituted2,19-(methyleneoxy)-androst-4-ene-3,17-dione (24).

To prepare the compound wherein X is ═CH₂, Scheme 8 is utilized.##STR9##

To the starting 17-keto compound (6), in EtOH, at 0° C., is added anexcess of NaBH₄. After 30 minutes the reaction is quenched with CH₃COCH₃ and concentrated. The residue is added to CH₂ C₁₂, washed with0.5N hydrochloric acid solution, water, then brine to yield thecorresponding 17-hydroxy compound (25). To this compound (25) in THF isadded aqueous hydrochloric acid solution to form the corresponding3-keto-17-hydroxy compound (26). This compound (26) is then treated with(C₆ H₅)₃ P═CH₂ to yield the corresponding 3-methylene-17-hydroxycompound (27). This compound is then oxidized at C-17 by Jones oxidationto form the 3-methylene-17-keto compound (28). Optionally, compound (28)may then be treated in a manner analogous to Scheme 2 to form thecorresponding 21-hydroxy-20-keto compound.

The following examples are provided to illustrate the present invention.They should not be construed as limiting it is any way.

EXAMPLE 1

To a stirred solution of 19-hydroxyandrost-4-ene-3,17-dione (1) (4.54 g,15.0 mmol) in CH₂ Cl₂ (40 ml) under argon atmosphere was addeddiisopropylethylamine (5.23 ml, 30.0 mmol) followed by1-chloro-2,5-dioxahexane (2.57 ml, 22.5 mmol). After 20 hours, thereaction was diluted with CH₂ Cl₂ (60 ml) and the organics were washedwith H₂ O (75 ml), 0.5N hydrochloric acid (2×75 ml), saturated NaHCO₃(35 ml), and brine (75 ml). Drying (MgSO₄) and concentration gave anorange oil (6.33 g). The oil was dissolved in 10 ml of EtOAc/hexane(65:35) and loaded onto a column. Flash chromatography (7.5×15 cm silicagel column), eluting with EtOAc/hexane (65:35) gave19-[(2-methoxyethoxy)methoxy]-androst-4-ene-3,17-dione (2). (Weight:4.44 g). HRMS calculated for C₂₃ H₃₄ O₅ (M+): 390.2406; found M+:390.2401; error =-1.3 ppm.

EXAMPLE 2

To a stirred solution of diisopropylamine (0.37 ml, 2.65 mmol) in THF (7ml) under argon and cooled to -20° C. was added n-BuLi (1.03 ml, 2.42Min hexane, 2.49 mmol). After 12 minutes, a cooled (-20° C.) solution oftrimethylchlorosilane (0.74 ml, 5.81 mmol) in THF (1 ml) was addedrapidly. After 2 minutes, a cooled (-20° C.) solution of the product ofExample 1 (2) (324 mg, 0.83 mmol) in THF (2 ml) was added dropwisefollowed by a 0.5 ml THF rinse. The reaction was stirred at -20° C. for30 minutes and then allowed to warm slowly to room temperature. Thereaction was stirred at room temperature for 30 minutes, triethylamine(1 ml) was added and the reaction was diluted to a 50 ml volume withethyl ether. The organics were washed with saturated NaHCO₃ (50 ml+20ml) followed by brine/saturated NaHCO₃ (20 ml of a 3:1 mixture). Drying(MgSO₄) and concentration gave a pale yellow oil. To this product wasadded hexane, the mixture concentrated, and then placed under highvacuum for 5 minutes to remove any remaining THF and triethylamine,yielding19-[(2-methoxyethoxy)methoxy]-3,17-bis-[(trimethylsilyl)oxy]androsta-2,4,16-triene(3) (quantitative).

EXAMPLE 3

To a stirred solution of the product of Example 2 (3) (0.83 mmol) in CH₂Cl₂ (8 ml) under argon and cooled to -20° C. was rapidly added a TiCl₄solution (2.49 ml of a 1M TiCl₄ in CH₂ Cl₂ solution, 2.49 mmol). A tansuspension resulted. Additional CH₂ Cl₂ (8 ml) was added. The reactionsuspension wasstirred at -20° C. for 35 minutes, diluted with CH₂ Cl₂and poured into saturated NaHCO₃. The layers were separated and theaqueous layer was extracted with Additional CH₂ Cl₂. The combinedorganics were washed with saturated NaHCO₃ (2×), 0.5N hydrochloric acid(1×), followed by brine. Drying (MgSO₄) and concentration gave a milkyoil. To this product was added 4 ml of EtOAc/hexane (50:50), the solidwas crushed, and the suspension was heatedwith a heat gun and thenallowed to cool to room temperature prior to loading the supernatantonto a flash column for chromatography (2×10cm silica gel column). Thesupernatant was loaded as stated, eluted with EtOAc/hexane (50:50), and15-20 ml fractions were collected. Concentrationof the productcontaining fractions gave a pale yellow oil. Et₂ O was added to theresidue and the flask was swirled to provide a solid. Concentration gavean oily, white solid (0.14 g). This product was then triturated with 2ml of Et₂ O/hexane (3:1). As much solid as possiblewas scraped from theside of the flask and the suspension was filtered to provide a whitesolid (56 mg). The solid was dried under high vacuum over refluxingacetone for 6 hours, yielding the compound of the formula below,mp204°-213° C.[3R-(3α,6aα,6bα,8aβ,11aα,-11bβ)]-3,4,6b,7,8,8a,10,11,11a,11b,12,13-dodecahydro-8a-methyl-6H-3,6a-methanocyclopenta[5,6]naphth[1,2-c]oxocin-2,9-dione,or alternatively named, 2,19-(methyleneoxy)androst-4-ene-3,17-dione.(Weight remained 56 mg.). ##STR10##

Elemental analysis: Calculated for C₂₀ H₂₆ O₃ : C, 76.40; H,8.34.Actual: C, 76.60; H, 8.53.

The corresponding sulfur compound, 2,19-(methylenethio)androst-4-ene-3,17-dione, was obtained in an analogous manner and meltedat 183°-199° C. This compound can also be named[3R-(3α,6aα,6bα,8aβ,11aα,11bβ)]-3,4,6b,7,8,8a,10,11,-11a,11b,12,13-dodecahydro-8a-methyl-6H-3,6a-methanocyclopenta[5,6]naphtho[1,2-c-]thiocin-2,9-dione.

EXAMPLE 4

The product of Example 3 was treated with a catalytic amount ofmethanesulfonic acid and an excess of ethylene glycol in solvent(benzene)and heated to reflux under Dean-Stark conditions to form thecorresponding 3,17-bis(ethylenedioxy)-5-ene compound (5).

EXAMPLE 5

A solution of the product of Example 4 in dichloromethane and t-butanolwastreated with 0.15% aqueous perchloric acid. The mixture was heated atgentle reflux for two hours with stirring and, then allowed to cool toroom temperature. The reaction mixture was then poured into saturatedsodium carbonate solution and extracted into EtOAc. The EtOAc extractwas washed with water and brine, dried over magnesium sulfate,concentrated, and chromatographed on silica gel eluting withEtOAc/hexane (2:3) to give the corresponding 17-one compound (6).

EXAMPLE 6

A solution of methyl methoxyacetate in tetrahydrofuran was slowly addedto a cold solution of lithium diisopropylamide, prepared fromdiisopropylamine and n-butyl lithium in hexane, in the same solvent. Asolution of the product of Example 5 in tetrahydrofuran was then addeddropwise over 5-10 minutes and the solution is stirred for three hoursat the same temperature. Saturated aqueous ammonium chloride solutionwas then added dropwise, and the mixture was poured into ice water andextracted with ethyl acetate. The extract was washed with brine, driedover sodium sulfate, filtered, and concentrated to afford the17-substituted steroid (7). The crude product was chromatographed onsilica gel, eluting with 1:1 ethyl acetate:hexane to afford the productasa mixture of isomers.

EXAMPLE 7

A solution of the product of Example 6 in pyridine and CH₂ Cl₂ waschilled to 0° C. and treated dropwise with thionyl chloride over 5-10minutes. After stirring for 75 minutes at the same temperature, thesolution was poured into ice water. The organic layer was washedtwicewith brine, dried over sodium sulfate, filtered and concentrated toafford crude product. Flash chromatography (30% ethyl acetate/70%hexane) afforded the methoxyester (8).

EXAMPLE 8

A solution of the product of Example 7 in toluene was chilled to -20° C.and treated dropwise with a 20% solution of diisobutylaluminum hydridein hexane. The solution was stirred at -20° C. for 30 minutes. Water wasadded and the mixture was stirredat 0° C. for 20 minutes, poured intoice water and extracted with 3:1 ether:dichloromethane. The extractswere washed with brine, dried oversodium sulfate, and concentrated. Theresidue was subjected to flash chromatography eluting with 3:2 ethylacetate:hexane to afford the alcohol(9).

EXAMPLE 9

To a solution of the product of Example 8 in THF was added 0.5N aqueousHCl. After 4 days, the reaction was diluted with CH₂ Cl₂ /H₂ O, thelayers separated, and the organics washed with brine, dried over sodiumsulfate, and concentrated. The hydroxyketone of the formula below wasisolated by flash chromatography (silica gel) eluting with EtOAc/hexane(4:1). HRMS: Calcd for C₂₂ H₃₁ O₄ : MH⁺ 359.2222; Found: MH⁺ 359.2204;Error: -5.0 ppm. ##STR11##

EXAMPLE 10

To a solution of the product of Example 4 (5) is addedm-chloroperbenzoic acid in methylene chloride at 0° C. The mixture ismaintained at 0° C. for 16 hours then diluted with methylene chlorideand washed with water, 10% sodium carbonate, and brine, then dried andevaporated. Chromatography gives the epoxide (11).

EXAMPLE 11

To a solution of the product of Example 10 (11) in THF and water isadded dropwise 70% aqueous perchloric acid and the reaction is stirredat room temperature for 48 hours. The mixture is diluted with methylenechloride, washed with aqueous Na₂ CO₃ and brine, then dried (MgSO₄) andconcentrated. Chromatography gives the corresponding diol (12).

EXAMPLE 12

To the product of Example 11 (12) in acetone at 0° C. is added dropwiseJones' reagent until a brown color persists for 15 minutes. The reactionis quenched with methanol. The mixture is then partitioned betweenmethylene chloride and water. The organic phase is washed with brine,and then dried and concentrated. Chromatography gives the hydroxyketone(13).

EXAMPLE 13

To the product of Example 12 (13) dissolved in benzene is added acatalyticamount of p-toluenesulfonic acid. The mixture is heated atreflux for 30 minutes using a Dean-Stark water trap. The cooled solutionis then poured into water. The organics are washed with aqueous Na₂ CO₃and brine, then dried and evaporated. The residue is chromatographed toaffordthe trione compound of the formula below,2,19-(methyleneoxy)androst-4-ene-3,6,17-trione (14). ##STR12##

EXAMPLE 14

To the product of Example 3 (4) in t-butyl alcohol was added chloranil(2.25 equivalents). The mixture was refluxed for 3.5 hours, cooled andfiltered and the filtrate concentrated. The residue was taken up inethyl acetate and washed with water, aqueous NaOH, and brine. Drying andconcentration, followed by chromatography afforded the compound[3R-(3α,6aα,6bα,8aβ,11aα,11bβ)]-3,4,6b,7,8,8a,10,11,11a,11b-decahydro-8a-methyl-6H-3,6a-methanocyclopenta[5,6]naphth[1,2-c]oxocin-2,9-dioneof the formula: ##STR13##

mp=193°-197° C.

¹ H NMR(CDCl₃): δ6.32 (dd, 1H, vinyl), 6.22 (dd, 1H, vinyl), 6.04 (s,1H, vinyl), 3.98 (ddd, 1H, 1/4 CH₂ OCH₂), 3.73 (dd, 1H, 1/4 CH₂ OCH₂),364 (d, 1H, 1/4 CH₂ OCH₂), 3.55 (dd, 1H, 1/4 CH₂ OCH₂), 0.94 (s, 3H,18--CH₃).

¹³ C NMR (CDCl₃): δ219.0, 201.2, 157.8, 138.2, 128.5, 127.9, 69.7, 67.1,49.1, 48.2, 47.8 44.2, 38.1 36.1, 35.5, 31.4, 21.3, 20.5, 13.6.

IR(KBr): 2956, 2854, 1740, 1662, 1616 cm⁻¹.

EXAMPLE 15

A suspension of sodium acetate in absolute chloroform containingformaldehyde dimethyl acetal and phosphoryl chloride is stirred atreflux for 1 hour. After addition of the product of Example 3 (4), themixture istreated dropwise with phosphoryl chloride over a period of 2.5hours. The reaction is subsequently stirred at reflux for theappropriate time. The suspension is allowed to cool and under vigorousstirring a saturated aqueous solution of sodium carbonate is addeddropwise until the pH of theaqueous layer becomes alkaline. The organiclayer is separated, washed withwater, and dried with sodium sulfate.After concentration and purification,the product obtained is thecompound of the formula: ##STR14##

What is claimed is:
 1. A method of inhibiting aromatase activity whichcomprises contacting an effective aromarase-inhibiting amount of acompound of the formula:wherein:----- represents a single or doublebond, A is O, S, SO, or SO₂, R is H, ═CH₂, ═O, or --OH, R¹ is H or C₁₋₄alkyl, R² is ═O, --OH, or --O--(C₁₋₄ alkanoyl), X is ═O, ═CH₂, --OH, or--O--(C₁₋₄ alkanoyl), and Y is H, --OH, or --O--(C₁₋₄ alkanoyl), andwhen Y═H, --OH, or --O--(C₁₋₄ alkanoyl), X may not include --OH, and Rmay not include ═O or --OH. with an aromatase enzyme.
 2. A methodaccording to claim 1 wherein the compound has the formula: ##STR15##wherein: ----- represents a single or double bond, and X, R, and R² aredefined as above.
 3. A method according to claim 1 wherein the compoundhas the formula: ##STR16## wherein: ----- represents a single or doublebond, and R,X, and Y are defined as above.
 4. A method according toclaim 1 wherein the compound is2,19-(methyleneoxy)androst-4-ene-3,17-dione and is represented by theformula: ##STR17##
 5. A method according to claim 1 wherein the compoundis 2,19-(methylenethio)androst-4-ene-3,17-dione and is represented bythe formula: ##STR18##
 6. A method according to claim 1 wherein thecompound is 2,19-(methyleneoxy)androsta-4,6-diene-3,17-dione and isrepresented by the formula: ##STR19##
 7. A method according to claim 1in which the aromatase inhibitor produces an anti-fertility effect.
 8. Amethod of treating hyperestrogenemia, which comprises administering to apatient having said condition an effective aromatase-inhibiting amountof a compound of the formula: ##STR20## wherein: ----- represents asingle or double bond,A is O, S, SO, or SO₂, R is H, ═CH₂, ═O, or --OH,R¹ is H or C₁₋₄ alkyl, R² is ═O, --OH, or --O--(C₁₋₄ alkanoyl), and X is═O, ═CH₂, --OH, or --O--(C₁₋₄ alkanoyl), and Y is H, --OH, or --O--(C₁₋₄alkanoyl), and when Y═H, --OH, or --O--(C₁₋₄ alkanoyl), X may notinclude --OH, and R may not include ═O or --OH.
 9. A method according toclaim 8 wherein the compound has the formula: ##STR21## wherein: -----represents a single or double bond, and X, R, and R² are defined asabove.
 10. A method according to claim 8 wherein the compound has theformula: ##STR22## wherein: ----- represents a single or double bond,and R,X, and Y are defined as above.
 11. A method according to claim 8wherein the compound is 2,19-(methyleneoxy)androst-4-ene-3,17-dione andis represented by the formula: ##STR23##
 12. A method according to claim8 wherein the compound is 2,19-(methylenethio)androst-4-ene-3,17-dioneand is represented by the formula: ##STR24##
 13. A method according toclaim 8 wherein the compound is2,19-(methyleneoxy)androsta-4,6-diene-3,17-dione and is represented bythe formula: ##STR25##
 14. A method of treating estrogen-induced orestrogen-stimulated disorders, which comprises administering to apatient having said condition an effective aromatase-inhibiting amountof a compound of the formula: ##STR26## wherein: ----- represents asingle or double bond,A is O, S, SO, or SO₂, R is H, ═CH₂, ═O, or --OH,R¹ is H or C₁₋₄ alkyl, R² is ═O, --OH, or --O--(C₁₋₄ alkanoyl), X is ═O,═CH₂, --OH, or --O--(C₁₋₄ alkanoyl), and Y is H, --OH, or --O--(C₁₋₄alkanoyl), and when Y═H, --OH, or -O-(C₁₋₄ alkanoyl), X may not include--OH, and R may not include =0 or --OH.
 15. A method according to claim14 wherein the compound has the formula: ##STR27## wherein: -----represents a single or double bond, and X, R, and R² are defined asabove.
 16. A method according to claim 14 wherein the compound has theformula: ##STR28## wherein: ----- represents a single or double bond,and R,X, and Y are defined as above.
 17. A method according to claim 14wherein the compound is 2,19-(methyleneoxy)androst-4-ene-3,17-dione andis represented by the formula: ##STR29##
 18. A method according to claim14 wherein the compound is 2,19-(methyleneoxy)androst-4-ene-3,17-dioneand is represented by the formula: ##STR30##
 19. A method according toclaim 14 wherein the compound is2,19-(methyleneoxy)androsta-4,6-diene-3,17-dione and is represented bythe formula: ##STR31##
 20. A method of treating hypertensive or edemousconditions, which comprises administering to a patient in need thereofan effective aromatase inhibiting amount of a compound of the formula:##STR32## wherein: ----- represents a single or double bond,A is O, S,SO, or SO₂, R is H, ═CH₂, ═O, or --OH, R¹ is H or C₁₋₄ alkyl, R² is ═O,--OH, or --O--(C₁₋₄ alkanoyl), X is ═O, ═CH₂, --OH, or --O--(C₁₋₄alkanoyl), and Y is H, --OH, or --O--(C₁₋₄ alkanoyl), and when Y═H,--OH, or --O--(C₁₋₄ alkanoyl), X may not include --OH, and R may notinclude ═O or --OH.
 21. A method according to claim 20 wherein thecompound has the formula: ##STR33## wherein: ----- represents a singleor double bond, and X, R, and R² are defined as above.
 22. A methodaccording to claim 20 wherein the compound has the formula: ##STR34##wherein: ----- represents a single or double bond, and R,X, and Y aredefined as above.
 23. A method according to claim 20 wherein thecompound is 2,19-(methyleneoxy)androst-4-ene-3,17-dione and isrepresented by the formula: ##STR35##
 24. A method according to claim 20wherein the compound is 2,19-(methylenethio)androst-4-ene-3,17-dione andis represented by the formula: ##STR36##
 25. A method according to claim20 wherein the compound is2,19-(methyleneoxy)androsta-4,6-diene-3,17-dione and is represented bythe formula: ##STR37##